CN101330928A

CN101330928A - Immunostimulatory combination for the prophylactics and treatment of hepatitis c

Info

Publication number: CN101330928A
Application number: CNA2006800457078A
Authority: CN
Inventors: A·扎巴莱塔阿兹皮罗茨; F·博拉斯库斯塔; J·普里拓瓦尔图纳; P·萨罗布乌加里扎; J·J·拉萨特萨加斯蒂贝尔扎
Original assignee: Proyecto de Biomedicina CIMA SL
Current assignee: Proyecto de Biomedicina CIMA SL
Priority date: 2005-10-07
Filing date: 2006-10-05
Publication date: 2008-12-24
Anticipated expiration: 2026-10-05
Also published as: SI1949913T1; DE602006014720D1; EP1949913A1; CA2625506A1; JP2009511452A; ATE469657T1; CN101330928B; HRP20100473T1; CA2625506C; ES2346570T3; EP1949913B1; WO2007042583A1; US20100047231A1; AU2006301171B9; PL1949913T3; PT1949913E; AU2006301171A1; AU2006301171B2; DK1949913T3; US20140056943A1

Abstract

The invention relates to an immunostimulatory combination for the prophylactics and treatment of hepatitis C. The inventive combination is characterised in that it comprises: a TLR3 agonist, a CD40 agonist and protein NS3 of the hepatitis C virus. The invention also relates to pharmaceutical compositions comprising the aforementioned immunostimulatory combination, to the use of said pharmaceutical compositions and to a kit comprising same. The invention further relates to a method of producing an immune response to the hepatitis C virus and to a vaccine against said virus.

Description

Be used to prevent and treat the immunostimulant combination of hepatitis C

Invention field

The present invention relates to a kind of immunostimulant combination that is used to prevent and treat hepatitis C, it comprises the NS3 albumen of HCV and can induce the special potent persistent CD8 of HCV virus ⁺And CD4 ⁺The adjuvant of replying.

Background of invention

According to estimates, the whole world has 100,000,000 7 thousand ten thousand people to infect hepatitis C virus, and the infection that this virus causes has now become the heavy burden of public health.And this sickness rate will continue constant in the coming years by inference.

HCV infects is characterized as the high tendentiousness that develops into chronic disease.HCV can renew work at 70% infected individual relaying, and wherein 20% can develop into liver cirrhosis and 2.5% and can develop into hepatocarcinoma.

Present reference treatment means are based on the therapeutic scheme that interferon uses.But these antiviral therapies cost an arm and a leg, and relative toxicity is more greatly and only effective to the patient that 50-60% receives treatment.Therefore be necessary and wish to research and develop new, more effective and have the therapeutic strategy of better toleration.

Nature (" Insights:Hepatitis C " .Nature 2005, Supplements; Vol.436, Nr.7053, pp 929-978) the up-to-date summary of couple HCV arranged in (" nature " (" seeing clearly: hepatitis C " nature 2005, supplementary issue, 436 volumes, 7053 phases, 929-978 page or leaf)).

Though it's a pity that we also do not find the effective vaccine to anti-hepatitis c virus, test data and phenomenon are thought us can find effective vaccine.Although antiviral antibody be to infect reply in synthetic, there is not cell toxicant-T cell (CD8 in being characterized as of this chronic process ⁺) and helper T cell (CD4 ⁺) cellullar immunologic response.So we infer that HCV has had the ability that its specificity invasion and attack antiviral immunity is replied, wherein cytotoxic T and auxiliary T intensity of replying and quality have determined that this patient whether can rehabilitation whether (rehabilitation or through treatment) automatically or they will be converted into chronic infection.

The main purpose of any vaccine all is a stimulator antigen specificity acquired immunity, and it is by B or T cell mediated.In this case, antigen presenting cell (APCs) especially activates in the T lymphocyte at the initiation specific immune response and has brought into play important function.APCs is mainly dendritic cells, they can be in the peripheral organ capture antigen and migrate to lymphatic organ after stimulating accepting to activate.There, dendritic cell is presented on their surfaces with the peptide prod (combine with the essence molecule of the main MHC of histocompatibility complex) after antigen (epi-position) degraded, and they produce chemotactic factor and cytokine simultaneously to attract and activation T-cell.MHC molecule (signal 1) is highly expressed in being characterized as of dendritic cell ciita process (being also referred to as maturation), and costimulator (signal 2) and polarization (polariser) cytokine be interleukin 12 (IL-12) (signal 3) for example.This maturation process is induced by for example pathogen component or these factors that often appear in inflammation or the cell injury process of host's molecule.The specific receptor of the product of these factors by deriving from microorganism acts on dendritic cell, for example the receptor (for example CD40) of TLR receptor (Toll-sample receptor), cytokine receptor (TNF-α, IL-1, IFN-α) or cell surface part.

APCs is to the stimulation of variety classes T-cell and activate the restriction that is subjected to the MHC molecule type on the one hand, is subjected to the restriction with the epi-position characteristic of MHC molecule forming composite on the other hand.For example, but some are accredited as specificity inducing cytotoxic CD8 ⁺The fragment of the activated virus protein of T lymphocyte (CTL) is usually referred to as lymphocyte epitopes or CD8 ⁺T cell epitope or CD8 ⁺Epi-position; Or but specificity is induced CD4 ⁺The activated epi-position of helper T lymphocyte (HTL) is usually referred to as CD4 ⁺Epi-position." HCV immunology data base " (http://hcv.lanl.gov/content/immuno/immuno-main.html) collected T lymphocyte (CD8 ⁺CTL and CD4 ⁺HTL) epi-position is that identify on the basis with the virus protein of hepatitis C virus different strains and chorista.

Therefore the exploitation of immunization protocol needs screening in advance to be fit to each individual peptide based on the use of polypeptide form epi-position, and this depends on the MHC molecule that each individuality presents.This shows that according to each individual MHC the necessary particular combinations of peptide of selecting could be as the epi-position under this condition.Use large-scale antigen to address this problem,, comprise CD8 because they are generally the poly epi-position and can have multiple epi-position in their sequence ⁺CTL and CD4 ⁺The epi-position of HTL, and the two can be presented by the MHC molecule of Different Individual.Thus, an independent antigen just can be used as vaccine in having the individuality of different MHC.

In the different proteins of HCV, core protein and NS3 albumen show intensive immunogenicity, can detect the potent CD8 at them in having overcome the crowd who infects ⁺CTL and CD4 ⁺The HTL immunne response.However, data with existing shows when core protein contacts with immune system cell also can produce illeffects to them, and this shows that using core protein is unadvisable as the antigen in the vaccine strategy.But on the other hand, NS3 albumen does not almost have the effect of this type, so NS3 albumen can be used as and induces CD8 ⁺CTL and CD4 ⁺The good candidate antigen of HTL immunne response.

CD4 ⁺HTL is at acquired immunity, activates at APC, CTL activates and memory is induced etc. in the mechanism of action and played a significant role.Document description HCV specific C D4 is especially arranged ⁺Cell is to keep antiviral CTL necessary (Grakoui A. etc., " HCV persistenceand immune evasion in the absence of memory T-cell help (not existing the HCV under the memory T accessory cell situation to continue to exist and immune evasion) "; Science, 2003; 302:659-662).Therefore, a kind of effective vaccine to anti-hepatitis c virus should provide CD8 ⁺CTL and CD4 ⁺The maximum that HTL replys is induced effectiveness.Therefore this vaccine need screen these immunne response specific antigens can be provided.

However, only depend on the effective vaccine that as if antigen makes up self can not provide at HCV.The maturation of considering dendritic cell is the activated essential condition of the effective initial sum of T lymphocyte, helps vaccine so add the immunostimulant combination of some adjuvants compositions, and this combination can stimulate the maturation of dendritic cell.The part of TLR receptor, the part of cytokine receptor or can be used as adjuvant, the perhaps synergistic combination of preferred these adjuvants at the part of the receptor of the intercellular part of having quoted.

For example, the immunostimulant combination that US2004/0141950 describes comprises the agonist of TLR agonist and tumor necrosis factor (TNF) or its receptor (TNFR) superfamily molecule, also can comprise a kind of antigen.In multiple possible combination, the combination table that the synthetic ligands of CD40 part (anti-CD 40 antibodies), poly (I:C) part, TLR3 forms reveals CD8 ⁺The lymphopoietic synergism of T.Similarly, (J.Exp.Med.2004 such as Ahonen; 199:775-784) provide relevant TLR/CD40 agonist to be independent of CD4 ⁺The lymphocytic mode inducing antigen of T specificity CD8 ⁺The data of the cooperative ability of CTL propagation and differentiation.Although these researchs have been described TLR/CD40 and have been activated CD8 ⁺The ability of T lymphocyte antigen specificity memory, still described research can not determine whether this TLR/CD40 agonist combination also can strengthen CD4 ⁺The HTL immunne response.

Under situation about being infected by HCV, the patient who contrasts infected patient and removed infection can find CD4 ⁺The HTL immunne response exists obviously different.However, though lower than the intensity of curing the patient, but in infected patient, detect CD8 ⁺The CTL immunne response.Therefore, though CTL is important effector lymphocyte colony in removing the HCV infection, CD4 ⁺Cell has also been brought into play important function in control disease.In addition, there is document description to induce CD4 ⁺The T lymphocyte is important (Grakoui A. etc., " HCV persistence and immune evasion in the absence ofmemory T-cell help (not existing the HCV under the memory T accessory cell situation to continue to exist and immune evasion) " for keeping antiviral CTL immunne response; Science, 2003; 302:659-662).These data show the immunity and the treatment of the viral disease that causes for HCV, induce CD8 ⁺And CD4 ⁺It is important that potent persistent antiviral immunity is replied.

Therefore, target of the present invention is that screening is applicable to the antigen of prevention and treatment hepatitis C and the immunostimulant combination that adjuvant is formed, and this combination can provide CD8 ⁺And CD4 ⁺The stimulation of immunne response, and make it more potent, complete and lasting.

Detailed Description Of The Invention

Primary goal of the present invention relates to the immunostimulant combination (below be called immunostimulant combination of the present invention) of prevention and treatment hepatitis C, wherein comprise TLR3 agonist, CD40 agonist or its DNA sequences encoding, and comprise the proteic polypeptide of NS3 of hepatitis C virus or have the CD8 of inducing ⁺And CD4 ⁺The described NS3 protein fragments of immunne response ability.

" TLR3 agonist " refers to make up or to combine and cause with TLR3 receptor (" toll sample receptor 3 ") part of cellullar immunologic response.TLR3 is the receptor of double-stranded RNA, but the double-stranded RNA conducted signal activates NF-κ B, generation I type interferon (IFN) (comprising IFN-α and IFN-β), and the latter can stimulate the maturation of dendritic cell.Lacking mice that TLR3 expresses reduces the immunne response of poly (I:C) (to HCV type virus replication time produce the similar TLR3 part of double-stranded RNA), lethal effect to poly (I:C) with D-galactosamine sensitization the time produces resistance, and the generation of inflammatory cytokine reduces (Alexopoulou etc., Nature, 2001, volume 413, page number 732-738).In specific embodiments of the present invention, described TLR3 part can be viral double-stranded RNA or polyinosinic acid-polycytidylicacid two strands (poly (I:C)).

" CD40 agonist " is meant the part that can make up or combine and cause cellullar immunologic response with the CD40 receptor.CD40 is a kind of molecule of expressing in the plasma membrane of dissimilar cells, for example bone-marrow-derived lymphocyte or antigen presenting cell (macrophage, dendritic cell etc.).Express in the T lymphocyte that the native ligand of CD40 (CD40L or CD154) mainly is activated after antigen recognition.The maturation that the CD40 that exists in CD40L and the antigen presenting cell interacts and induces CD40.This phenomenon causes antigen presenting cell to have stronger induce immune response ability to be similar to the mode that the pathogen source stimulates.Therefore, the CD40 agonist in the immunostimulant combination of the present invention refers to the CD40L part on the one hand, has referred to keep the CD40L fragment of CD40 binding ability and inducing cell or immunne response on the other hand.In a specific embodiments, this part can be that the specific antibody (anti-CD40) of CD40 or its have kept the fragment of CD40 binding ability.In addition, CD40 part or its fragment can protein form in immunostimulant combination or recombinant nucleic acid (DNA) form of coding part exist, for example be used for the viral vector of transfection or gene therapy.

" antigen " be meant any can be in individual organism induce immune response (comprising humoral immunization and cellular immunization) or can be when contacting with immunocyte the material of inducing cell immunne response (breed, activate and/or the immunocyte that accelerates the ripening, produce cytokine or antibody).Particularly, antigen can be the recombinant protein of the fragment of virus protein, peptide or described virus protein, described virus protein or the synthetic polypeptide of energy induce immune response.

" CD8 ⁺Induce epi-position " be meant and can induce CD8 by specificity ⁺Activated antigenic fragment of cytotoxic T lymphocyte (CTL) or part of polypeptide chain." CD4 ⁺Induce epi-position " be meant and can induce CD4 by specificity ⁺The fragment of the activated antigenic part of polypeptide chain of helper T lymphocyte (HTL).

" NS3 albumen " is meant the non-structural protein NS3 of hepatitis C virus, it is that a kind of molecular weight is the albumen of 67kDa, contain 2 domains, one is to comprise 189 amino acid whose serine-proteolytic enzymes of N-terminal, and another is to contain 442 amino acid whose territories with helicase-atpase activity of C-terminal.The proteic sequence of NS3 that comprises in the immunostimulant of the present invention combination polypeptide can conform to chorista with the arbitrary strain system of hepatitis C virus.In a specific embodiments, can obtain to contain the proteic polypeptide of NS3 by recombinant technique.In a concrete non-limiting embodiments of the present invention, the sequence that reorganization NS3 albumen uses is SEQ ID.NO:1 (referring to Genebank accession number DQ068198.1 and AAY84763.1, VRL 28-NOV-2005).We have also used another recombiant protein sequence SEQ ID.NO:2 (referring to Genebank accession number D90208).

In another optional embodiment of the present invention, also can use the polypeptide that contains the NS3 protein fragments, described fragment can be induced CD4 ⁺And CD8 ⁺Immunne response.Therefore, described fragment must contain at least one CD8 ⁺Induce epi-position and a CD4 ⁺Induce epi-position.

In a specific embodiments, immunostimulant combination of the present invention comprises poly (I:C), anti-CD 40 antibodies and contains the proteic polypeptide of NS3.

In a preferred embodiment of the present invention, immunostimulant combination comprises that all can form the component of an identical Pharmaceutical composition part, but component is the pharmacy receiving amount in wherein every.In addition, the present invention also relates to described Pharmaceutical composition.

In another specific embodiments of the present invention, the component of immunostimulant combination can form the part of at least two kinds of Pharmaceutical compositions.Similarly, the present invention relates to the purposes of described immunostimulant combination, it is characterized in that giving simultaneously described Pharmaceutical composition.In another embodiment of the invention, described immunostimulant combined feature is to give described Pharmaceutical composition at different time by identical or different route of administration.So a specific embodiments of the present invention relates to the medicine box that is used to give above-mentioned immunostimulant combination, it is characterized by and comprise Pharmaceutical compositions different at least two.

On the other hand, the present invention relates to produce method, it is characterized by the immunostimulant that comprises the above definition that gives effective dose and make up with induce immune response at the hepatitis C virus immunne response.In a preferred embodiment, the inventive method comprises preventative processing.In a preferred embodiment, the inventive method comprises therapeutic treatment.

At last, the present invention also relates to vaccine to anti-hepatitis c virus, it is characterized by the immunostimulant combination that comprises above definition from and reach target of the present invention.

The accompanying drawing summary

Fig. 1. with anti-CD40, poly (I:C) with NS3 albumen immune induction multi-epitope CD4 ⁺And CD8 ⁺T replys.Give HHD mice (two every group) the anti-CD40 of lumbar injection 50g.After four hours, give their intravenous injection 50g polies (I:C) and lumbar injection 500g reorganization NS3 albumen (SEQ.ID.NO:1).After six days, put to death animal and extract the analysis that splenocyte is used for different antigenic stimulated in vitro and institute's induce immune response.(A) under the situation that IL-2 exists, use epi-

position CD8

⁺1073,1406 or 1038 (10 μ M) irritation cell.Then, in each group splenocyte, detect loading (polypeptide; The black bar shaped) or not load (contrast; The white bar shaped) solubilizing reaction of the target cell of corresponding polypeptide.The gained result is with the effector lymphocyte: target cell is that 100: 1 ratio shows.(B) in an identical manner splenocyte is cultivated with polypeptide 1073 (black circle), 1406 (white triangles) or 1038 (the black triangles) of variable concentrations (0.1-10 μ M), stimulated and get culture supernatant after 48 hours, detect the content of IFN-γ with the ELISA method.(C) the NS3 albumen (SEQ.ID.NO:3) produced with the antibacterial of 1 μ g/ml of the NS3 albumen (SEQ.ID.NO:1) that uses in the immunity with 5 or 1 μ g/ml again or stimulated splenocyte 48 hours with culture medium (contrast) is with detection CD4 ⁺Reply.After this time period, collect supernatant and use the ELISA method to detect the generation of IFN-γ.

Fig. 2. detect and with poly (I:C) and anti-CD40 immunity the time, induce CD4 ⁺And CD8 ⁺The proteic necessary amounts of the NS3 that T replys.With poly (I:C) and the immune HHD mice of anti-CD40 (two every group), its scheme as described in Figure 1 with NS3 albumen (SEQ ID.NO:1) (500,250,125 or 25 μ g/ mice).Also comprise a matched group with the same procedure immunity, its use 5 μ g NS3 (SEQ.ID.NO:1), 50

μ g polypeptide

1073 and 1038 as antigen with poly (I:C) and anti-CD40 immunity.After six days, put to death animal and extract splenocyte with different antigenic stimulus (A).For detecting inductive solubilizing reaction, use epi-position CD8 ⁺1073 (10 μ M) and IL-2 irritation cell 5 days.Afterwards, the target cell incubation with the loading polypeptide of not commensurability effector lymphocyte and fixed amount detects this reaction.In addition, analyze inductive CD8 by measuring IFN-γ generation ⁺Reply.For this reason, use polypeptide 1073 (B) and 1038 (C) irritation cell of variable concentrations.Also use NS3 albumen (SEQ.ID.NO:1) (D) irritation cell with quantitative CD4 ⁺Reply.Detect the content of IFN-γ in the supernatant after 48 hours.

Fig. 3. together during having other strain mice of different MHCs induce CD4 with anti-CD 40 with NS3 albumen with poly (I:C) ⁺And CD8 ⁺Reply.The scheme of pointing out according to Fig. 1 with 100 μ g NS3 (SEQ.ID.NO:1) with poly (I:C) and the immune C57BL6 mice of anti-CD 40 (the MHC molecule is the H-2b type) (two every group) once (white box) or twice (black square).After six days, put to death animal and with splenocyte and synantigen incubation not to detect derivative CD4 ⁺And CD8 ⁺Reply.(SEQ.ID.NO:7) stimulate splenocyte and detect CD8 with restriction epi-position H-2Db1629-1637 (GAVQNEVTL) ⁺Reply (A).NS3 albumen (SEQ.ID.NO:1) is (B) as measuring CD4 ⁺The stimulus object of replying.After incubation two days later, collect supernatant and measure the generation of IFN-γ.

Fig. 4 .NS3 albumen with poly (I:C) but and anti-CD 40 induce the CD8 of recognition expression HCV albuminous cell ⁺Reply.As shown in Figure 1, give (A) HHD mice (two every group) injection 100 μ g albumen (SEQ.ID.NO:2) and poly (I:C) and anti-CD 40.After six days, use the T1/HCVcon cell handled through mitomycin (transfection the proteic T1 cell of expression of HCV) to stimulate their splenocyte with sacrifice of animal and in the presence of IL-2.After stimulation in five days, in detecting, lytic activity measures the ability of this splenocyte identification T1/HCVcon cell.In this detection, with T1 control cells (white circular) incubation of the splenocyte of varying number and T1/HCVcon cell of fixed qty (black circle) or untransfected.

Fig. 5. induce T CD4 with poly (I:C) and anti-CD 40 with NS3 albumen ⁺And CD8 ⁺Reply.As shown in Figure 1, give HHD mice (two every group) injection 100 μ gNS3 albumen (SEQ.ID.NO:2) and poly (I:C) and anti-CD 40.After two weeks, secondary immunity animal under identical condition.After the secondary immunity six days sacrifice of animal and the splenocyte that extracts them are used to study CD8 ⁺And CD4 ⁺The persistency that T replys.(A) use epi-position CD8 ⁺1073 (10 μ M) or stimulate splenocyte not containing under the antigenic situation collect the culture fluid supernatant to detect the growing amount of IFN-γ after 48 hours.(B) with this splenocyte with epi-position 1073 (10 μ M) and IL-2 incubation 5 days, study the ability that their dissolvings load the target cell of polypeptide 1073 then.For this reason, with the loading polypeptide 1073 (black circles) of the effector lymphocyte of varying number and fixed qty or do not load the target cell incubation of polypeptide (white circle).(C) by (SEQ.ID.NO:2) or not existing under the antigenic situation stimulating this splenocyte research CD4 with NS3 albumen (1 μ g/ml) ⁺Reply.After 48 hours, collect supernatant and detect the growing amount of IFN-γ.

The embodiment of specific embodiments of the present invention

Following examples are intended to show the specific embodiments that forms present patent application, do not have any restricted.

Associated materials and method

Epi-position, antigen and reagent

Use Fmoc chemistry (Wellings DA.and Atherton E.MethodsEnzymol 1997; 289:44-67) employed peptide of multistep peptide synthesizer synthetic or epi-position.With the Kaiser ninhydrin test each step is monitored.When end of synthesis, its montage is also used the trifluoroacetic acid deprotection, wash with Anaesthetie Ether.Detect the peptide purity of any time gained all greater than 90% through HPLC.

The peptide and the epi-position of synthesizing and using among table 1. embodiment

Peptide or epi-position	Sequence
Peptide or epi-position	Sequence	1038-1047	GLLGCIITSL；SEQ.ID.NO：4
1073-1081	CVNGVCWTV；SEQ.ID.NO：5	1038-1047	GLLGCIITSL；SEQ.ID.NO：4
1073-1081	CVNGVCWTV；SEQ.ID.NO：5	1406-1415	KLVALGINAV；SEQ.ID.NO：6
1629-1637	GAVQNEVTL；SEQ.ID.NO：7	1406-1415	KLVALGINAV；SEQ.ID.NO：6

The numbering of peptide or epi-position refers to its relative HCVH position, with sufficient sequence in the human C type hepatitis H strain that often is used as prototype as with reference to (GeneBank accession number M67463).Therefore, for example data base " HCV immunology data base " (http://hcv.lanl.gov/content/immuno/immuno-main.html) has collected T-lymphocyte (comprising cytotoxic t-lymphocyte and the auxiliary T-lymphocyte) epi-position that does not identify in the virus protein of homophyletic system and chorista with hepatitis C virus, all sorts according to the relative position that is with respect to this virus H strain that provides among the GeneBank.

Make 655 amino acid whose recombinant polypeptides of apparatus as immunogen, it contains NS3 albumen (SEQ.ID.NO:1; Genebank accession number AAY84763.1, VRL 28-NOV-2005; 631 aminoacid) sufficient sequence.Except proteic 631 aminoacid of NS3, this polypeptide also contains the c-myc sequence and the histidine afterbody that are used to detect the monoclonal antibody of anti--myc.Can use and produce this albumen in the pichia pastoris phaff.It can suspend and be kept in the solution that contains Tris 22.5mM, carbamide 3.76M, NaCl 300mM.Can use this albumen of nickel column chromatography purification.

Another kind of recombinant polypeptide also is used as immunogen, and it contains 635 aminoacid, has comprised NS3 albumen (SEQ.ID.NO:2; Genebank accession number D90208) sufficient sequence.Except the aminoacid corresponding with NS3, this polypeptide also contains the histidine afterbody that is useful on purification.Can obtain the corresponding DNA sequences with NS3 by Sal I and Not I digested plasmid gWIZ, this plasmid contains NS3 sequence (by Dr.G.Inchauspe, Lyon, France provides).Plasmid pET-45 ( ⁺) this digestion product of clone between the BsrG I of (Novagen, Madison WI) and the Not I site.This product can be expressed in escherichia coli (E.Coli), uses nickel post affinity chromatography to use ion exchange chromatography to be purified then earlier.

Similar, use a kind of recombinant polypeptide (Mikrogen in the vitro detection; Catalog number 94302) as antigen, it contains 20 aminoacid at non-structural protein NS2 end and 508 initial aminoacid of NS3 albumen (SEQ.ID.NO:3) of HCV.

Use available from the poly (I:C) (catalog number 27-4732-01) of Amersham as the TLR3 agonist.

Use anti-CD 40 antibodies as the CD40 agonist, purification is from hybridoma FGK-45 (Rolink A. etc., immune 1996.5:319-330)

Molten born of the same parents' thing QCL-1000 detects through king crab ameboid cell (Bio Whittaker), and the endotoxic content of all reagent is all less than 1 unit/mg product.

Mice

Six to eight all big C57B1/6 mices are available from Harlan.Also use the HHD mice, it contains people HLA-A2.1 (Pascolo S.et al., J.Exp.Med.1997.185:2043-2051) transgenic of molecule.All animals are all raised under anosis old terms, and according to the predetermined processing of this mechanism.

Cell line

Use T2 cell (Salter R. etc. immunogenetics, 1985 21:235-246) as the target cell of chromium release assay, use cytotoxic t-lymphocyte (CTL) in the mensuration from the HHD mice.

The T1 cell (T1/HCVcon cell) of HCV coding region vector plasmid that used transfection, the identification that is used for the expression of HCV albuminous cell detects.These cells are by doctor D.Moradpour (Freiburg, Germany; Volk B. etc., J Gen Virol.2005; 86:1737-1746) provide.The T1 cell (ATCC, catalog number .CRL-1991) of use untransfected in contrast.

All growths (RPMI 164010% calf serum, 100U/ml penicillin, 100 μ g/ml streptomycins, 2mM glutamine and 50 μ M 2 mercapto ethanols) in complete medium of all cells.The culture medium of T1/HCVcon system also contains 2mg/ml of G418 (Gibco).

Immunity

With two groups of mices of 50 μ g anti-CD 40 abdominal channels immunity.After four hours, inject 50 μ g poly (I:C) (vein) and not commensurability antigens: the mixture (abdominal cavity) of NS3 albumen or NS3 albumen and peptide to them.

Stimulate splenocyte to produce cytokine

Splenocyte is resuspended in complete medium and is containing or do not containing under peptide or the proteic condition of recombinant HCV NS3 with 8 * 10 ⁵Cells/well adds the 0.2ml culture fluid in 96 orifice plates at the bottom of the U-shape.

Collect supernatant two days later, detect the existence of IFN-γ according to manufacturer's operation instruction with ELISA (BD-Pharmingen).

Detect the lytic activity of CTL

Reply in order to detect CTL, will be from splenocyte and 37 ℃ of incubation 2h of peptide (10 μ M) of immunized animal, washed twice and with 7.5 * 10 ⁶Cells/well adds the 24-orifice plate.In the experiment that detects identification T1/HCVcon cell, with 7.5 * 10 ⁶HDD mouse boosting cell and organizine rimocidin C (Sigma) before handled 7.5 * 10 ⁵The T1/HCVcon cell is cultivated together.In all cases, (Boehringer-Mannheim GmbH Germany), reclaims cell to carry out chromium-release test after 5 days to add 2.5U/ml IL-2 to each hole two days later.

Exist or do not exist under the situation of peptide (target), the T2 target cell incubation that effector lymphocyte and 3000 of varying number have been loaded 51Cr 4 hours.Under situation, with effector lymphocyte and the T1/HCVcon or the T1 incubation that load 51Cr with the T1/HCVcon irritation cell.Incubation is collected the culture supernatant after 4 hours.

Calculate SL percentage ratio according to following formula: (the spontaneous cpm of experiment cpm-)/(the spontaneous cpm of maximum cpm-) * 100, wherein spontaneous dissolving (measured value is recorded as spontaneous cpm) is corresponding with the target cell of incubation under no effect cell condition, obtains maximum dissolving (maximum cpm) with 5%Tritonx100 incubation target cell.

Embodiment 1

Anti-CD 40 and poly (I:C) are with the CD4 of the multiple epi-position of NS3 albumen immune induction ⁺And CD8 ⁺T replys.

Itself demonstrate them with anti-CD40 and poly (I:C) immunity and can represent CD8 by using ⁺The synthetic polypeptide of cell epitope is induced CD8 very effectively as immunogen ⁺Reply.Though this method can be induced intensive replying, studies show that, when with inducing CD4 ⁺When the low dosage NS3 albumen of replying (5 μ g/ mice) was immune together, it improved CD8 ⁺The intensity of replying, and can strengthen high affine CD8 ⁺Reply (also can discern low concentration is antigenic replys).In addition, HLA molecule effectively individual that only those and selected epi-position is had same restrictions with the peptide immunity.In order to solve this 2 point, people have carried out research to determine whether can not only induce CD4 with the immunity that more heavy dose of reorganization NS3 albumen carries out ⁺Reply also and can induce CD8 ⁺Reply.For this reason, with anti-poly (I:C) and CD40 immune mouse, study inductive replying with NS3 then.So, give the anti-CD40 of HHD mice (two every group) lumbar injection 50 μ g.After four hours, give their intravenous injections 50 μ g polies (I:C) and lumbar injection 500 μ g reorganization NS3 albumen (SEQ.ID.NO:1).After six days, put to death mice and extract splenocyte.Tire for analyzing NS3, when using poly (I:C)+anti-CD40 to induce CD8 ⁺And CD4 ⁺When T replys with not synantigen stimulated in vitro splenocyte that can special activation splenocyte group.(A) for analyzing CD8 ⁺Reply, in first test, under the condition that IL-2 exists, stimulated splenocyte 5 days with epi-position CD8+1073,1406 or 1038.Then, each the group cell for stimulating through peptide detects the ability that their dissolvings have loaded the target cell (black bar shaped) of corresponding peptides or do not loaded the contrast target cell (white bar shaped) of peptide.Figure 1A has shown the effector lymphocyte: target cell is 100: 1 resulting results.(B) also by studying at identical CD8 ⁺Inductive CD8 after the IFN-γ generation analysis NS3 immunity of epi-position ⁺Reply.For this reason, cultivate with splenocyte with 1073 (black circles), 1406 (white triangles), 1038 (black triangle) of various dose.Cultivate after 48 hours, collect supernatant, detect the content of IFN-γ.(C) for analyzing inductive CD4 ⁺Reply, stimulate splenocyte with the NS3 albumen (SEQ.ID.NO:1) that uses in the immunity.Also bacterial origin NS3 albumen (SEQ.ID.NO:3) irritation cell of commodity in useization.With with more preceding identical method, detect the activation degree by IFN-γ generation.

At first, this antigen can be detected and CD8 can be induced ⁺Reply, this can and induce IFN-γ to produce method (Figure 1B) and detect by chromium release assay method (Figure 1A).In addition, this is replied is multiple epi-position, can corresponding multiple CD8 ⁺Epi-position, and the feature that the NS3 sequence has comprised these epi-positions is (for example:

polypeptide

1073,1406 and 1038).At last, determine that it also can induce CD4 ⁺Reply CD4 ⁺Reply and to discern the NS3 albumen that uses in the immunity and the NS3 albumen (Fig. 1 C) of commercialization bacterial origin.Lower to replying of the latter, may be because there are differences between two kinds of albumen sequences and the albumen of bacterial expression shorter, make it lack number of C D4 ⁺The discernible epi-position of T lymphocyte.

Embodiment 2

NS3 is enough to induce CD4 with poly (I:C) and anti-CD40 administration with 25 μ g reorganization ⁺And CD8 ⁺T replys.

We can learn that 5 μ g NS3 can induce CD4 from before test ⁺Reply and can not induce CD8 ⁺Reply, so we wish to find and are enough to induce CD8 ⁺The NS3 lowest dose level of replying.For this reason, with the immune HHD mice of 500,250,125,25 μ g NS3 (SEQ.ID.NO:1).Also comprise and use CD8 ⁺The corresponding polypeptide of epi-position (can be induced CD8 ⁺Replying) additional 5 μ g NS3 (SEQ.ID.NO:1) (can induce CD4 ⁺Reply) immune matched group.For this reason, in each treated animal, analyze its CD8 with the NS3 immunity of a dosage ⁺Reply and CD4 ⁺Reply.Can load epi-position CD8 by dissolving ⁺The ability of 1073 target cell (Fig. 2 A) reaches variable concentrations epi-position CD8 ⁺1073 (Fig. 2 B) and 1038 (Fig. 2 C) produce the ability of IFN-γ and analyze CD8 ⁺Reply.Detect CD4 by the ability that variable concentrations NS3 (SEQ.ID.NO:1) is produced IFN-γ ⁺Reply.This test shows that all detected NS3 dosage all can be induced CD8 ⁺Reaction, in the dissolved cell repercussion study of polypeptide 1073 (Fig. 2 A), the dosage of 25 μ g can be induced replying of weak degrees.And all dosage all can be induced the generation to the IFN-γ of epi-position 1073 (Fig. 2 B) and 1038 (Fig. 2 C), even this shows that dosage reduces the ability that still can keep it to induce multi-epitope to reply.At last, as our expection, they all can induce CD4 ⁺Reaction.Consider in most of the cases, use 25 μ g NS3 can reduce inductive replying, thus after test in select the dosage of 100 μ g/ mices, begin not observe from this dosage and induce the enhancing of replying.

Embodiment 3

Poly (I:C) is induced CD4 with anti-CD 40 with other strain mice that NS3 albumen immunity together has different MHC ⁺And CD8 ⁺Reply

If an antigen is the same with NS3 albumen big, it might exist can be by the CD4 of the different molecular submission of MHC ⁺And CD8 ⁺Epi-position, we have studied this immunization protocol and induced CD4 in having other strain mice of different MHC ⁺And CD8 ⁺The ability of replying.For this reason, the C57/B16 mice that has H-2b restriction MHC molecule with 100 μ g NS3 (SEQ.ID.NO:1) immunity.Reply in order to strengthen this, one group of mice is accepted single immunization, and another winding is subjected to the secondary booster immunization.At first the generation by anti-peptide 1629-1637 (SEQ.ID.NO:7) IFN-γ detects CD8 ⁺Reply, this peptide contains can be by the CD8 of I H-2 Db class MHC molecule submission ⁺Epi-position.As seen from Figure 3, in two groups of mices, all induced detectable replying, be significantly higher than the once animal groups (white square) of immunity of only acceptance although accept the animal groups level of replying of twice immunity (black square).Generation by anti-reorganization NS3 albumen (SEQ.ID.NO:1) detects CD4 ⁺Reply, the result shows that also replying of twice immunity (black square) is better than single immunization (white square).

Embodiment 4

NS3 albumen with poly (I:C) but and anti-CD 40 induce the CD8 of recognition expression HCV albuminous cell ⁺Reply

Use NS3 albumen may kill the cell response that is infected by HCV in order to study with poly (I:C) and immune whether can the inducing of anti-CD 40, but the use transfection target cell (T1/HCVcon) external model of the proteic plasmid of expression of HCV.These cellular expressions belong to the identical peptide of I class MHC molecule, the same with the cellular expression that is infected by HCV; Therefore, any the replying at them can be assumed to replying at the latter.The NS3 albumen (SEQ.ID.NO:1) that the experiment of Fig. 1-3 is used is corresponding to the different Strain of Strain in a kind of and the T1/HCVcon cell.These two kinds of strain systems and the CD8 that is studied so far ⁺Epi-position is different.In order to optimize to CD8 in the T1/HCVcon cell ⁺The identification ability of epi-position uses NS3 albumen (SEQ.ID.NO:2) as immunogen in this experiment, and its sequence is higher than the albumen homology in the T1/HCVcon cell.With after the 100 μ gNS3 immunity HHD mice six days, with T1/HCVcon cytositimulation mouse boosting cell.In detecting, lytic activity analyzes identification ability to the T1/HCVcon cell.For this reason, with T1/HCVcon cell and T1 control cells and the splenocyte incubation that is stimulated.As shown in Figure 4, the ability (white circle) of replying the proteic T1 cell of dissolving expression of HCV with the NS3 immune induction is stronger than the ability (black circles) of cracking T1 control cells.

Embodiment 5

Poly (I:C) and anti-CD 40 are with NS3 albumen immune induction persistence T CD4 ⁺And CD8 ⁺Reply

One of prerequisite main character of a kind of immunization protocol is an inducing sustained immune response, thereby the protection that immunity is brought can continue the long period.Whether can induce such to reply in order to study anti-CD 40 and poly (I:C) with NS3 albumen, according to the scheme of describing among the embodiment 1 with 100 μ g NS3 protein immunization HDD mices.In order to strengthen this reaction, under identical condition, carry out booster immunization after 15 days to animal.Secondary immunity is put to death animal and continued at that time with analysis with their splenocyte of different antigenic stimulus after six days CD8 ⁺And CD4 ⁺T replys.In order to study CD8 ⁺T replys, with epi-position 1073 irritation cells and detect generation and the lytic activity of IFN-γ.Shown in Fig. 5 A, secondary immunity can produce a large amount of IFN-γ, but not exist antigen then can not after six days when stinging menstruating on time after pregnancy anti-CD 40s and poly (I:C) with NS3 albumen mice immunized splenocyte with peptide 1073.And these cells can dissolve the target cell (black circles) that stimulates through peptide 1073 and can not dissolve and do not contain antigenic target cell (white circle) (Fig. 5 B).At last, study CD4 with the NS3 albumen that uses in the immunity as antigen ⁺Reply.Fig. 5 shows that this immunization protocol also can induce the CD4 of potent lasting and specific recognition NS3 ⁺Reply.

Sequence table

SEQUENCE LISTING

<110>PROYECTO DE BIOMEDICINA CIMA S.L.

<120>IMMUNO-STIMULANT COMBINATION FOR PROPHYLAXIS AND TREATMENT OF

HEPATITIS C

<130>05009

<160>7

<170>PatentIn Version3.1

<210>SEQ.ID.NO.：1

<211>631

<212>PRT

＜213〉hepatitis C virus

<220>

<221>MISC_FEATURE

＜223〉unstructuredness NS3 albumen

<400>1

Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly Cys

1 5 10 15

Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu

20 25 30

Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala Thr Cys Ile

35 40 45

Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Lys Thr Ile

50 55 60

Ala Ser Ser Lys Gly Pro Val Ile Gln Met Tyr Thr Asn Val Asp Gln

65 70 75 80

Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ala Arg Ser Leu Thr Pro

85 90 95

Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp

100 105 110

Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser

115 120 125

Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu

130 135 140

Cys Pro Ala Val His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr

145 150 155 160

Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Gly Leu Glu

165 170 175

Thr Thr Met Arg Ser Pro Val Phe Ser Asp Asn Ser Ser Pro Pro Ala

180 185 190

Val Pro Gln Ser Tyr Gln Val Ala His Leu His Ala Pro Thr Gly Ser

195 200 205

Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys

210 215 220

Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala

225 230 235 240

Tyr Met Ser Lys Ala His Gly Ile Asp Pro Ile Ile Arg Thr Gly Val

245 250 255

Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys

260 265 270

Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile

275 280 285

Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Asp

290 295 300

Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Thr Val Leu

305 310 315 320

Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile

325 330 335

Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys

340 345 350

Ala Ile Pro Leu Glu Ala Ile Lys Gly Gly Arg His Leu Ile Phe Cys

355 360 365

His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu

370 375 380

Gly Val Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile

385 390 395 400

Pro Ala Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr

405 410 415

Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Ash Thr Cys Val

420 425 430

Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr

435 440 445

Thr Thr Leu Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg

450 455 460

Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu

465 470 475 480

Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp

485 490 495

Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg

500 505 510

Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His

515 520 525

Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala

530 535 540

His Phe Leu Ser Gln Thr Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu

545 550 555 560

Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro

565 570 575

Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu

580 585 590

His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu

595 600 605

Ile Thr Leu Thr His Pro Ile Thr Lys Tyr Ile Met Thr Cys Met Ser

610 615 620

Ala Asp Leu Glu Val Val Thr

625 630

<210>SEQ.ID.NO.：2

<211>635

<212>PRT

＜213〉hepatitis C virus

<220>

<221>MISC_FEATURE

＜223〉unstructuredness NS3 albumen

<400>2

Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly Cys

1 5 10 15

Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Asp Gly Glu

20 25 30

Val Gln Val Leu Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr Cys Val

35 40 45

Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Ser Lys Thr Leu

50 55 60

Ala Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln

65 70 75 80

Asp Leu Val Gly Trp Pro Ala Pro Pro Gly Ala Arg Ser Met Thr Pro

85 90 95

Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp

100 105 110

Val Val Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser

115 120 125

Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu

130 135 140

Cys Pro Ser Gly His Val Val Gly Ile Phe Arg Ala Ala Val Cys Thr

145 150 155 160

Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Ser Met Glu

165 170 175

Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala

180 185 190

Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser

195 200 205

Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys

210 215 220

Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala

225 230 235 240

Tyr Met Ser Lys Ala His Gly Ile Glu Pro Asn Ile Arg Thr Gly Val

245 250 255

Arg Thr Ile Thr Thr Gly Gly Pro Ile Thr Tyr Ser Thr Tyr Cys Lys

260 265 270

Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile

275 280 285

Cys Asp Glu Cys His Ser Thr Asp Ser Thr Thr Ile Leu Gly Ile Gly

290 295 300

Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu

305 310 315 320

Ala Thr Ala Thr Pro Pro Gly Ser Ile Thr Val Pro His Pro Asn Ile

325 330 335

Glu Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe Tyr Gly Lys

340 345 350

Ala Ile Pro Ile Glu Ala Ile Lys Gly Gly Arg His Leu Ile Phe Cys

355 360 365

His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Thr Gly Leu

370 375 380

Gly Leu Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile

385 390 395 400

Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr

405 410 415

Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val

420 425 430

Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr

435 440 445

Thr Thr Leu Pro Gln Asp Ala Val Ser Arg Ala Gln Arg Arg Gly Arg

450 455 460

Thr Gly Arg Gly Arg Ser Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu

465 470 475 480

Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp

485 490 495

Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val Arg

500 505 510

Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His

515 520 525

Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp Ala

530 535 540

His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Leu Pro Tyr Leu

545 550 555 560

Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro

565 570 575

Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu

580 585 590

His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu

595 600 605

Val Thr Leu Thr His Pro Ile Thr Lys Tyr Ile Met Ala Cys Met Ser

610 615 620

Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val

625 630 635

<210>SEQ.ID.NO.：3

<211>528

<212>PRT

＜213〉artificial sequence

<220>

＜223〉derive from the proteic chimeric reorganization of hepatitis C virus NS2 and NS3

<400>3

Gly Arg Glu Ile Leu Leu Gly Pro Ala Asp Gly Met Ala Ser Lys Gly

1 5 10 15

Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly

20 25 30

Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln

35 40 45

Val Glu Gly Glu Val Gln Ile Val Pro Thr Ala Ala Gln Thr Phe Leu

50 55 60

Ala Thr Cys Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly

65 70 75 80

Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Ser

85 90 95

Asn Val Asp Lys Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ser Arg

100 105 110

Ser Leu Ala Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr

115 120 125

Lys His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly

130 135 140

Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly

145 150 155 160

Gly Pro Leu Leu Cys Pro Val Gly His Ala Val Gly Ile Phe Arg Ala

165 170 175

Ala Val Cys Thr Arg Gly Val Ala Lys Ala Ala Asp Phe Ile Pro Val

180 185 190

Glu Asn Leu Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser

195 200 205

Ser Pro Pro Val Val Pro Gln Ser Phe Gln Val Ala His Leu His Ala

210 215 220

Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala

225 230 235 240

Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu

245 250 255

Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile

260 265 270

Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr Ser

275 280 285

Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ala Gly Gly Ala Tyr

2902 95 300

Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile

305 310 315 320

Leu Gly Ile Gly Thr Val Leu Asp Gln Gly Glu Thr Ala Gly Ala Lys

325 330 335

Leu Val Val Phe Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro

340 345 350

His Pro Asn Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro

355 360 365

Phe Tyr Gly Lys Ala Ile Pro Leu Glu Val Ile Lys Gly Gly Arg His

370 375 380

Leu Ile Phe Cys His Ser Lys Arg Lys Cys Asp Glu Leu Ala Thr Lys

385 390 395 400

Leu Val Ala Met Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp

405 410 415

Val Ser Val Ile Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp

420 425 430

Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys

435 440 445

Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe

450 455 460

Thr Ile Glu Thr Thr Thr Leu Pro Gln Asp Ala Val Ser Arg Thr Gln

465 470 475 480

Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val

485 490 495

Ala Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys

500 505 510

Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu

515 520 525

<210>SEQ.ID.NO.：4

<211>10

<212>PRT

＜213〉hepatitis C virus

<220>

<221>misc_feature

＜223〉corresponding to the epi-position 1038-1047 of NS3 virus protein

<400>4

Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu

1 5 10

<210>SEQ.ID.NO.：5

<211>9

<212>PRT

＜213〉hepatitis C virus

<220>

<221>misc_feature

＜223〉corresponding to the epi-position 1073-1081 of NS3 virus protein

<400>5

Cys Val Asn Gly Val Cys Trp Thr Val

1 5

<210>SEQ.ID.N0.：6

<211>10

<212>PRT

＜213〉hepatitis C virus

<220>

<221>misc_feature

＜223〉corresponding to the epi-position 1406-1415 of virus protein

<400>6

Lys Leu Val Ala Leu Gly Ile Asn Ala Val

l 5 10

<210>SEQ.ID.NO.：7

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>7

Gly Ala Val Gln Asn Glu Val Thr Leu

1 5

Claims

1. immunostimulant combination that is used to prevent and treat hepatitis C is characterized in that comprising:

A) TLR3 agonist,

B) DNA sequence of CD40 agonist or this agonist of encoding, and

C) comprise the proteic polypeptide of hepatitis C virus NS, or have the CD8 of inducing ⁺And CD4 ⁺The proteic fragment of described NS3 of responsibility.

2. the immunostimulant of claim 1 combination is characterized in that described TLR3 agonist is poly (I:C).

3. claim 1 or 2 immunostimulant combination is characterized in that described CD40 agonist is selected from anti-CD 40 antibodies, CD40L and the above fragment that has kept they and CD40 binding ability.

4. the immunostimulant of claim 3 combination is characterized in that described CD40 agonist is a kind of anti-CD 40 antibodies.

5. the immunostimulant of claim 1 combination is characterized in that comprising:

A) poly (I:C),

B) anti-CD 40 antibodies, and

C) contain the proteic polypeptide of NS3.

6. claim 4 or 5 immunostimulant combination is characterized in that the proteic polypeptide of the described NS3 of containing is the polypeptide of a kind of SEQ of having ID.NO:1.

7. each immunostimulant combination in the claim 1 to 6 is characterized in that all components forms the part of single Pharmaceutical composition.

8. each immunostimulant combination in the claim 1 to 6 is characterized in that all components can form the part of at least two kinds of different Pharmaceutical compositions.

9. the purposes of the immunostimulant of claim 8 combination is characterized in that giving simultaneously described Pharmaceutical composition.

10. the purposes of the immunostimulant of claim 9 combination is characterized in that giving described Pharmaceutical composition at different time.

11. the purposes of the immunostimulant of claim 9 combination is characterized in that giving described Pharmaceutical composition by different way of administration.

12. a Pharmaceutical composition contains the immunostimulant combination of each description in the claim 1 to 8, it is characterized in that each component all exists with pharmaceutically acceptable amount.

13. a medicine box is used for giving the immunostimulant combination of each description of claim 1 to 6, it is characterized in that containing at least two kinds of different Pharmaceutical compositions.

14. the method to hepatitis C virus generation immunne response is characterized in that comprising the stimulant combination that gives each definition in the claim 1 to 8 with effective dose, with induce immune response.

15. the method for claim 14 is characterized in that comprising preventative processing.

16. the method for claim 14 is characterized in that comprising therapeutic treatment.

17. the vaccine to anti-hepatitis c virus is characterized in that comprising the immunostimulant combination of each definition in the claim 1 to 8.